Throughout this application various publications are referred to within parenthesis. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications, in their entireties, are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
Sonic hedgehog signaling controls the differentiation of motor neurons in the ventral neural tube but the intervening steps are poorly understood. A differential screen of a cDNA library derived from a single Shh-induced motor neuron has identified a novel homeobox gene, MNR2, expressed by motor neuron progenitors and transiently by post-mitotic motor neurons. The ectopic expression of MNR2 in neural cells initiates a program of somatic motor neuron differentiation characterized by the expression of homeodomain proteins, by neurotransmitter phenotype and by axonal trajectory. Our results suggest that the Shh-mediated induction of a single transcription factor, MNR2, is sufficient to direct somatic motor neuron differentiation.
The assembly of neural circuits in the vertebrate central nervous system (CNS) is initiated by the generation of distinct classes of neurons at characteristic positions. The specification of neuronal identity in the CNS appears to be controlled by inductive signals secreted by embryonic organizing centers (Lumsden and Krumlauf, 1996; Tanabe and Jessell, 1996). These signals appear to define neuronal fates by regulating the expression of cell-intrinsic determinants, many of which are transcription factors (Bang and Goulding, 1996). However, the pathways by which specific inductive signals determine the fate of individual neuronal cell types in the CNS are poorly defined. As a consequence, it is unclear whether there are individual transcription factors assigned, in a dedicated manner, to the specification of particular neuronal subtypes or whether the parallel actions of several factors are required.
Spinal motor neurons constitute one subclass of CNS neuron for which some early differentiation steps have been defined (Pfaff and Kintner, 1998). The differentiation of motor neurons depends on spatial signals provided by Sonic Hedgehog (Shh) secreted from the notochord and floor plate (Marti et al. 1995; Roelink et al., 1995; Tanabe et al., 1995; Chiang et al. 1996; Ericson et al. 1996). Shh acts initially to convert medial neural plate cells into a population of ventral progenitors (Ericson et al., 1996) and later directs the differentiation of ventral progenitors into motor neurons and interneurons at distinct concentration thresholds (Roelink et al. 1995; Ericson et al., 1997). The Shh-induced pathway of motor neuron differentiation appears, however, to operate within the context of an independent program of neurogenesis. Neural progenitors that have been exposed to Shh undergo two or more cell divisions before leaving the cell cycle and acquiring motor neuron properties (Ericson et al., 1996). Over this period, ventral progenitors require continued Shh signaling, achieving Shh-independence and committing to a motor neuron fate only late in their final division cycle (Ericson et al., 1996).
Cells in the ventral neural tube respond to graded Shh signaling with the establishment of distinct ventral progenitor populations defined by the expression of the homeodomain proteins Pax6 and Nkx2.2 (Ericson et al., 1997). These two progenitor populations generate distinct classes of motor neurons. Pax6+ progenitors give rise to somatic motor neurons whereas Nkx2.2+ progenitors generate visceral motor neurons (Ericson et al. 1997). As these two progenitor populations leave the cell cycle they express different homeodomain proteins that characterize distinct motor neuron subtypes (Tsuchida et al., 1994; Varela-Echavarria et al., 1996; Ericson et al., 1997; Pattyn et al. 1997). The activity of Pax6 is necessary for the differentiation of somatic motor neurons within the hindbrain (Ericson et al., 1997; Osumi et al., 1997) but it appears that its function is indirect, being required to repress the expression of Nkx2.2 (Ericson et al., 1997).
The dispensibility of Pax6 for somatic motor neuron generation implies the existence of additional genes that determine somatic motor neuron identity. Moreover, the late commitment of progenitors to a somatic motor neuron fate suggests that the onset of expression of such genes occurs only during the final division cycle of motor neuron progenitors. To identify such determinants a screen for genes expressed by somatic motor neuron progenitors was performed and described, here is the characterization of a novel homeobox gene, MNR2.
MNR2 is expressed selectively by Pax6+ motor neuron progenitors and persists transiently in post-mitotic somatic motor neurons. The ectopic expression of MNR2 in vivo is sufficient to activate a program of somatic motor neuron differentiation characterized by the expression of several homeodomain proteins and Choline Acetyltransferase (ChAT), by the autoactivation of MNR2 and by the extension of axons into ventral roots. This program of motor neuron differentiation is accompanied by the repression of spinal interneuron fates. Thus, the Shh-triggered differentiation of ventral progenitor cells into somatic motor neurons may be directed by the expression of a single homeodomain protein, MNR2.
This invention provides an isolated nucleic molecule encoding a motor neuron restricted MNR2, protein that has the capacity to induce motor neuron generation.
This invention provides an isolated nucleic molecule encoding a motor neuron restricted pattern, MNR2, protein.
This invention provides a vector which comprises the isolated nucleic acid encoding a motor neuron restricted pattern, MNR2, protein.
This invention provides a host cell containing the vector which comprises the isolated nucleic acid encoding a motor neuron restricted pattern, MNR2, protein.
This invention provides a method of producing a polypeptide having the biological activity of a mammalian MNR2 which comprises growing host cells selected from a group consisting of bacterial, plant, insect or mammalian cell, under suitable conditions permitting production of the polypeptide.
This invention provides an isolated nucleic acid molecule of at least 15 contiguous nucleotides capable of specifically hybridizing with a unique sequence included within the sequence of the nucleic acid molecule encoding a motor neuron restricted pattern, MNR2, protein.
This invention provides an antisense oligonucleotide having a nucleic acid sequence capable of specifically hybridizing to an mRNA molecule encoding a MNR2 protein.
This invention provides a monoclonal antibody directed to an epitope of an MNR2 protein.
This invention provides a purified MNR2 protein.
This invention provides a method of inducing differentiation somatic motor neurons which comprises expressing MNR2 protein in neural progenitor cells.
This invention provides a transgenic nonhuman mammal which comprises an isolated nucleic acid encoding a motor neuron restricted pattern, MNR2, protein, is a DNA molecule.
This invention provides a method of determining physiological effects of expressing varying levels of MNR2 protein in a transgenic nonhuman mammal which comprises producing a panel of transgenic nonhuman animals, each a transgenic nonhuman mammal, each nonhuman mammal expressing a different amount of MNR2 protein.
This invention provides a method of producing an isolated purified MNR2 protein which comprises: a) inserting a nucleic acid molecule encoding a MNR2 protein into a suitable vector; b) introducing the resulting vector into a suitable host cell; c) selecting the introduced host cell for the expression of the MNR2 protein; d) culturing the selected cell to produce the MNR2 protein; and e) recovering the MNR2 protein produced.
This invention provides a method of inducing differentiation of somatic motor neurons in a subject comprising administering to the subject the purified MNR2 protein in an amount effective to induce differentiation of somatic motor neurons in the subject.
This invention provides a pharmaceutical composition comprising a purified MNR2 protein and a pharmaceutically acceptable carrier.
This invention provides a method for treating a subject afflicted with an abnormality associated with a lack of one or more normally functioning motor neurons which comprises introducing an amount of a pharmaceutical composition comprising a purified MNR2 protein and a pharmaceutically acceptable carrier effective to generate somatic motor neurons from undifferentiated motor neuron precursor cells in the subject, thereby treating the subject afflicted with the abnormality associated with the lack of one or more normally functioning motor neurons.
This invention provides a method of treating a subject afflicted with a neurodegenerative disease which comprises introducing an amount of a pharmaceutical composition which comprises a purified MNR2 protein and a pharmaceutically acceptable carrier effective to generate somatic motor neurons from undifferentiated precursor motor neuron cells in the subject, thereby treating the subject afflicted with the neurodegenerative disease.
This invention provides for a method of treating a subject afflicted with an acute nervous system injury which comprises introducing an amount of a pharmaceutical composition which comprises a purified MNR2 protein and a pharmaceutically acceptable carrier effective to generate motor neurons from undifferentiated precursor motor neuron cells in a subject, thereby treating the subject afflicted with the acute nervous system injury.
This invention provides for a method of treating a subject afflicted with an acute nervous system injury, wherein the acute nervous system injury is localized to a specific central axon which comprises surgical implantation of a pharmaceutical composition comprising a MNR2 protein and a pharmaceutically acceptable carrier effective to generate motor neurons from undifferentiated precursor motor neuron cells located proximal to the specific central axon, so as to alleviate the acute nervous system injury localized to a specific central axon, thereby treating the subject afflicted with the acute nervous system injury.
This invention provides a method for diagnosing a chronic neurodegenerative disease associated with the expression of a MNR2 protein in a sample from a subject which comprises: (a) obtaining DNA from the sample of the subject suffering from the chronic neurodegenerative disease; (b) performing a restriction digest of the DNA with a panel of restriction enzymes; (c) separating the resulting DNA fragments by size fractionation; (d) contacting the resulting DNA fragments with a nucleic acid probe capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a MNR2 protein, wherein the sequence of a nucleic acid molecule encoding a MNR2 protein is linked at a specific break point to a specified nucleic acid sequence and labeled with a detectable marker; (e) detecting labeled bands which have hybridized to the nucleic acid probe capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a MNR2 protein, wherein the sequence of a nucleic acid molecule encoding a MNR2 protein is linked at a specific break point to a specified nucleic acid sequence to create a unique band pattern specific to the DNA of subjects suffering from the chronic neurodegenerative disease; (f) preparing DNA obtained from a sample of a subject for diagnosis by steps (a-e); and (g) comparing the detected band pattern specific to the DNA obtained from a sample of subjects suffering from the chronic neurodegenerative disease from step (e) and the DNA obtained from a sample of the subject for diagnosis from step (f) to determine whether the patterns are the same or different and to diagnose thereby predisposition to the chronic neurodegenerative disease if the patterns are the same.
This invention provides a method for diagnosing a chronic neurodegenerative disease associated with the expression of a MNR2 protein in a sample from a subject which comprises: (a) obtaining RNA from the sample of the subject suffering from chronic neurodegenerative disease; (b) separating the RNA sample by size fractionation; (c) contacting the resulting RNA species with a nucleic acid probe capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a MNR2 protein, wherein the sequence of a nucleic acid molecule encoding a MNR2 protein is linked at a specific break point to a specified nucleic acid sequence and labeled with a detectable marker; (d) detecting labeled bands which have hybridized to the RNA species to create a unique band pattern specific to the RNA of subjects suffering from the chronic neurodegenerative disease; (e) preparing RNA obtained from a sample of a subject for diagnosis by steps (a-d); and (f) comparing the detected band pattern specific to the RNA obtained from a sample of subjects suffering from the chronic neurodegenerative disease from step (d) and the RNA obtained from a sample of the subject for diagnosis from step (f) to determine whether the patterns are the same or different and to diagnose thereby predisposition to the chronic neurodegenerative disease if the patterns are the same.
This invention provides a functionally equivalent analog of MNR2 that induces MNR2 differentiation of neural progenitor cells.
This invention provides a functionally equivalent analog of MNR2 that prevents MNR2 differentiation of neural progenitor cells.
This invention provides a method of treating a subject afflicted with a neuromuscular disease which comprises introducing an amount of a pharmaceutical composition comprising a purified MNR2 protein and a pharmaceutically acceptable carrier effective to activate acetylcholine to activate muscle cells.